Determination method for in-vitro degradation rate of xyloglucan and derivative thereof

Abstract

The invention discloses a method for determining the in-vitro degradation rate of xyloglucan and derivatives thereof, and belongs to the technical field of biological material detection. According to the method, a hydrogen peroxide system is combined with a precipitator method, so that efficient separation of xyloglucan degradation and undegraded substances is realized, and the problem that degradation products are difficult to separate in a traditional method is solved; a hydrogen peroxide remover is introduced, so that the problem of color development distortion caused by oxidation of hydrogen peroxide on phenol by a sulfuric acid method is solved, and the applicability obstacle of a traditional detection method in an oxidation system is solved; the determination method is simple to operate, does not need large expensive equipment, is less in interference, and realizes the unification of detection precision and operation simplicity and convenience. The innovative technology provides a precise regulation and control means for the fields of drug delivery, tissue engineering, wound dressings and the like, and the performance and clinical application value of related products can be remarkably improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomaterial detection, and particularly relates to a method for determining the in-vitro degradation rate of xyloglucan and derivatives thereof. BACKGROUND

[0002] Xyloglucan is a natural polysaccharide composed of a β-1,4-glucan backbone and α-xylose side chains, which has excellent biocompatibility, controllable gelation ability and biodegradability, and is widely used in drug delivery systems, tissue engineering scaffolds and wound dressings. Since the in-vivo degradation behavior of xyloglucan and its derivative materials directly affects the functional timeliness and biological safety, it is of great significance to guide the application of xyloglucan in vivo to establish an in-vitro degradation method that can simulate the in-vivo degradation microenvironment and realize reliable detection of the degradation rate.

[0003] At present, the degradation methods of xyloglucan mainly include enzymatic hydrolysis, physical method and chemical oxidation method. Although the enzymatic hydrolysis method has strong specificity, it is difficult to simulate the degradation behavior under physiological conditions due to the lack of corresponding enzymes in the human body. The physical method destroys the polysaccharide structure through ultrasonic crushing, γ-ray or microwave assistance, which is simple and green, but has high energy consumption, low degradation efficiency and may destroy the ordered structure of the material. The chemical oxidation method uses strong oxidizing agents such as strong acid, sodium periodate or Fenton reagent to efficiently break the sugar chain structure, but the reaction is violent, the by-products may have cytotoxicity, and the degradation degree is difficult to control accurately. Moreover, the existing methods generally have the problems of difficulty in separation of degradation products or complexity of subsequent purification. Therefore, it is of great significance to develop a new degradation method with low cost, high efficiency and controllable degradation performance to expand the application of xyloglucan materials in the fields of biomedicine, food packaging and the like.

[0004] In the detection and analysis of degradation products, the existing technology also faces many challenges. The commonly used detection methods reported at present include weight loss method, high performance liquid chromatography (HPLC) and gel permeation chromatography (GPC). The weight loss method is simple to operate, but has low sensitivity and long experimental period, and is not suitable for soluble materials. Although the HPLC and GPC methods have high accuracy, they are complex in pretreatment and expensive in equipment. The phenol sulfuric acid method has the advantages of high sensitivity, strong anti-interference ability, simple operation and low cost, and is an ideal choice for quantitative analysis of sugars. However, the reducing phenol is easily oxidized, which leads to distortion of the color reaction, so it cannot be directly applied to the oxidation degradation system. SUMMARY

[0005] The application provides a method for determining the in-vitro degradation rate of xyloglucan or its derivatives, comprising the following steps: The wood glucomannan or wood glucomannan derivative is added to a hydrogen peroxide system for degradation; then the degradation solution is taken and a hydrogen peroxide remover is added for reaction; after the reaction is completed, centrifugation is performed, and the supernatant is divided into two parts, which are recorded as a degradation group and a total sugar group, respectively; a precipitant is added to the supernatant of the degradation group, and water is added to the supernatant of the total sugar group, and then centrifugation is performed after mixing uniformly, and the supernatant of each is taken; the supernatant is placed in an ice water bath, phenol is added, mixed uniformly, and then concentrated sulfuric acid is added, mixed uniformly; then it is placed in a boiling water bath for reaction, and after the reaction is completed, the reaction solution is taken out and cooled with an ice water bath; the absorbance of the reaction solution of the degradation group and the total sugar group is determined; and the degradation rate is determined.

[0006] In the above determination method, the wood glucomannan derivative is one or more of enzymatically modified wood glucomannan, carboxymethylated wood glucomannan, acetylated wood glucomannan, and oxidized wood glucomannan.

[0007] In the above determination method, the hydrogen peroxide system is a hydrogen peroxide-water solution, a hydrogen peroxide-phosphate buffer solution, a hydrogen peroxide-sodium chloride solution, or a hydrogen peroxide-carbonate buffer solution; and the mass concentration of hydrogen peroxide in the hydrogen peroxide system is 1-15%, preferably 3-10%.

[0008] In the above determination method, the mass-volume ratio of the wood glucomannan or wood glucomannan derivative to the hydrogen peroxide system is 1:1500-1:200, g:mL; preferably 1:1000-1:250, g:mL.

[0009] In the above determination method, the degradation conditions are water bath shaking at 35-39°C; preferably water bath shaking at 37°C.

[0010] In the above determination method, the hydrogen peroxide remover is manganese dioxide or a catalase solution; when the hydrogen peroxide remover is manganese dioxide, the mass-volume ratio of the manganese dioxide to the degradation solution is 1:1-20:1, mg:mL; when the hydrogen peroxide remover is a catalase solution, the volume ratio of the catalase solution to the degradation solution is 1:1-1:10; and the concentration of the catalase solution is 0.1-0.5 mg / mL.

[0011] In the above determination method, the precipitant is one or more of anhydrous ethanol, isopropyl alcohol, acetone, n-butanol, or ethyl acetate; and the amount of the precipitant is 1-10 times, preferably 3-5 times, the volume of the supernatant.

[0012] In the above determination method, the amount of water added to the total sugar group is consistent with the amount of precipitant added to the degradation group.

[0013] In the above determination method, the absorbance is determined at 484 nm.

[0014] In the above determination method, the degradation rate is the ratio of the absorbance value of the supernatant of the degradation group to the absorbance value of the supernatant of the total sugar group.

[0015] This invention provides the application of the above-described assay method in drug delivery, tissue engineering, and wound dressing development.

[0016] The beneficial effects of this invention are as follows: This invention achieves a significant breakthrough in the in vitro degradation detection of xyloglucan and its derivatives through innovative degradation system design and optimized detection methods. Regarding the degradation system, an H₂O₂ solution is used as the degradation system, and the separation of degraded and undegraded products can be achieved by combining it with a precipitant method. This system more closely resembles the in vivo oxidation microenvironment and exhibits good biocompatibility. In terms of the detection method, this invention creatively introduces a hydrogen peroxide removal agent, completely eliminating the colorimetric reaction deviation caused by the direct oxidation of phenol by H₂O₂, thus achieving highly sensitive and accurate quantitative analysis of the degradation products.

[0017] Hydrogen peroxide exhibits levels comparable to reactive oxygen species at wound sites. Furthermore, compared to other reactive oxygen species reagents, hydrogen peroxide demonstrates higher stability, allowing it to penetrate sample membranes and tissues more easily and remain in samples for a longer period. Therefore, the hydrogen peroxide system used in this invention more closely resembles the in vivo oxidative microenvironment.

[0018] The xyloglucan degradation detection technology proposed in this invention demonstrates significant application value in the biomedical field. For example, in drug sustained-release systems, by accurately measuring the degradation kinetics of xyloglucan carriers, the release curve of controlled-release drugs can be optimized, achieving drug release at the expected rate, significantly improving therapeutic efficacy and reducing side effects. In tissue engineering, this technology can be used to establish a matching model between the degradation rate of scaffold materials and cell growth rate, assessing the stability of materials in oxidative environments, thereby accelerating the development of biomaterials such as biomimetic cartilage. In the development of chronic wound dressings, this invention can precisely control the degradation rate of dressings in a simulated wound microenvironment, maintaining their structural integrity while promoting wound healing, reducing dressing change frequency, and lowering the risk of infection. This innovative technology provides precise control methods for drug delivery, tissue engineering, and wound dressings, significantly improving the performance and clinical application value of related products.

[0019] This invention employs a hydrogen peroxide system combined with a precipitant method to achieve highly efficient separation of degraded and undegraded products, overcoming the difficulty in separating degradation products in traditional methods. The introduction of a hydrogen peroxide removal agent eliminates the color distortion caused by hydrogen peroxide oxidation of phenol in the sulfuric acid method, overcoming the applicability limitations of traditional detection methods in oxidative systems. This detection method is simple to operate, requires no large or expensive equipment, and has minimal interference, achieving a balance between detection accuracy and ease of operation. This innovative technology provides precise control methods for drug delivery, tissue engineering, and wound dressings, significantly improving the performance and clinical application value of related products. Attached Figure Description

[0020] Figure 1 The state of xyloglucan in a hydrogen peroxide-water degradation system (Example 1).

[0021] Figure 2 The image shows the reaction state of hydrogen peroxide and the removal agent in the degradation system; the left side shows the addition of manganese dioxide (Example 1), and the right side shows the addition of catalase (Example 7).

[0022] Figure 3 The images show the state of hydrogen peroxide after reaction with the removal agent and centrifugation; the left side shows the manganese dioxide precipitate after centrifugation (Example 1), and the right side shows the catalase precipitate after centrifugation (Example 7).

[0023] Figure 4 The state of the undegraded sample after centrifugation with the addition of a precipitant to the supernatant (Example 1). Detailed Implementation

[0024] In this invention, xyloglucan is derived from Shanghai Maclean Biochemical Technology Co., Ltd.

[0025] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0026] Example 1 The degradation of xyloglucan and the determination of its degradation rate are performed as follows: Weigh 0.025 g xyloglucan and place it in 10 mL of hydrogen peroxide-water solution (H2O2 mass concentration 5%). Degrade it by shaking in a water bath at 37 °C for 7 d, 14 d, 28 d, 42 d and 60 d respectively.

[0027] Then take 2 mL of degradation solution, add 0.032 g of manganese dioxide (removal agent), react for 15 min, centrifuge at 10000 rpm for 10 min; divide the supernatant into two portions, namely the degradation group and the total sugar group, 1 mL each; Add 4 mL of anhydrous ethanol (precipitant) to the degradation group, mix well, centrifuge at 10000 rpm for 10 min, and take 2 mL of supernatant; add 4 mL of water to the total sugar group, mix well, centrifuge at 10000 rpm for 10 min, and take 2 mL of supernatant; take 2 mL of water as a blank control group and label it tube 0. Place the supernatant (including tube 0) in an ice-water bath, slowly add 5 mL of phenol aqueous solution (5% by mass), mix well, then slowly add 5 mL of concentrated sulfuric acid (98% concentration), mix well; heat in a boiling water bath for 20 min, then remove and cool in an ice-water bath. Using tube 0 as a blank, the absorbance of the degradation group and the total sugar group at 484 nm was measured. The ratio of the absorbance value of the degradation group to the absorbance value of the total sugar group is the in vitro degradation rate of xyloglucan.

[0028] In the above experiment, three parallel sampling points were used. Since the total sugar concentration was the same in the system at different degradation times, it was not necessary to test the total sugar content at each sampling point. The 7-day results could be used to represent the total sugar content at different degradation times. The measurement results are shown in Table 1.

[0029] Table 1 Results of xyloglucan degradation rate determination Example 2 In this embodiment, the in vitro degradation rate of xyloglucan was determined; the degradation system was a hydrogen peroxide-water solution with a H2O2 mass concentration of 3%; the precipitant was isopropanol; other steps were the same as in Example 1, and the results are shown in Table 2.

[0030] Table 2 Results of xyloglucan degradation rate determination Example 3 In this embodiment, the in vitro degradation rate of xyloglucan was determined; the degradation system was hydrogen peroxide-phosphate buffer, the mass concentration of H2O2 was 10%; the precipitant was acetone; other steps were the same as in Example 1, and the results are shown in Table 3.

[0031] Table 3 Results of xyloglucan degradation rate determination Example 4 This embodiment measures the in vitro degradation rate of acetylated xyloglucan (prepared according to the following literature: Acetylation modification improved the physicochemical properties of xyloglucan from tamarind seeds. International Journal of Biological Macromolecules, 2022, 223, 193-201.). The sample amount of acetylated xyloglucan was 0.04 g; the degradation system was a hydrogen peroxide-sodium chloride solution with a H2O2 mass concentration of 5% and a sodium chloride mass concentration of 0.85%; the precipitant was n-butanol; other steps were the same as in Example 1, and the results are shown in Table 4.

[0032] Table 4. Degradation rate of acetylated xylo-glucan Example 5 This embodiment measures the in vitro degradation rate of carboxymethylated xyloglucan (prepared according to the following literature: Synthesis and Characterization of Superhigh Moisturizing Carboxymethyl Tamarind Xyloglucan and Its Potential Application in Cosmetics. Biomacromolecules 2025, 26, 1647−1658.). The sample amount of carboxymethylated xyloglucan was 0.04 g; the degradation system was hydrogen peroxide-phosphate buffer with a H2O2 mass concentration of 5%; the precipitant was ethyl acetate; other steps were the same as in Example 1, and the results are shown in Table 5.

[0033] Table 5. Degradation rate of carboxymethylated xylo-glucan Example 6 This embodiment measures the in vitro degradation rate of enzymatically hydrolyzed modified xyloglucan (prepared according to Example 1 of patent CN 106362222 A); wherein the sample amount of enzymatically hydrolyzed modified xyloglucan is 0.01 g; the degradation system is hydrogen peroxide-phosphate buffer, the mass concentration of H2O2 is 5%; the precipitant is anhydrous ethanol; other steps are the same as in Example 1, and the results are shown in Table 6.

[0034] Table 6. Degradation rate of enzymatically modified xyloglucan Example 7 In this embodiment, the in vitro degradation rate of xyloglucan was determined; catalase was used as the hydrogen peroxide removal agent, the concentration of catalase solution was 0.1 mg / mL, and the volume ratio of catalase to degradation solution was 1:5; other steps were the same as in Example 1, and the results are shown in Table 7.

[0035] Table 7 Results of xyloglucan degradation rate determination Comparative Example 1 This comparative study validated the effect of the presence or absence of the removal agent (manganese dioxide / catalase) on the in vitro degradation rate of xyloglucan; the degradation rate of xyloglucan was measured only at a degradation time of 7 days, and compared with that of Examples 1 and 7. The results are shown in Table 8.

[0036] Table 8 Results of xyloglucan degradation rate determination after 7 days The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for determining the in vitro degradation rate of xyloglucan or its derivatives, characterized in that, Includes the following steps: Xyloglucan or xyloglucan derivatives were added to a hydrogen peroxide system for degradation. The degradation solution was then collected, and a hydrogen peroxide removal agent was added to initiate the reaction. After the reaction was complete, the mixture was centrifuged, and the supernatant was divided into two portions, designated as the degradation group and the total sugar group. A precipitant was added to the supernatant of the degradation group, and water was added to the supernatant of the total sugar group. After mixing thoroughly, the mixture was centrifuged, and the supernatant of each group was collected. The supernatant was placed in an ice-water bath, phenol was added, and the mixture was stirred. Then, concentrated sulfuric acid was added and stirred. The mixture was then placed in a boiling water bath for further reaction. After the reaction was complete, the reaction solution was removed and cooled in an ice-water bath. The absorbance of the degradation group and the total sugar group reaction solution was measured, and the degradation rate was determined.

2. The determination method according to claim 1, characterized in that, The xyloglucan derivative is one or more of the following: enzymatically modified xyloglucan, carboxymethylated xyloglucan, acetylated xyloglucan, and oxidized xyloglucan.

3. The determination method according to claim 1, characterized in that, The hydrogen peroxide system is a hydrogen peroxide-aqueous solution, a hydrogen peroxide-phosphate buffer solution, a hydrogen peroxide-sodium chloride solution, or a hydrogen peroxide-carbonate buffer solution.

4. The determination method according to claim 1, characterized in that, The mass-to-volume ratio of the xyloglucan or xyloglucan derivative to the hydrogen peroxide system is 1:1500 to 1:200, g:mL.

5. The determination method according to claim 1, characterized in that, The degradation conditions are: water bath agitation degradation at 35~39℃.

6. The determination method according to claim 1, characterized in that, The hydrogen peroxide removal agent is manganese dioxide or catalase solution.

7. The determination method according to claim 1, characterized in that, The precipitant is one or more of anhydrous ethanol, isopropanol, acetone, n-butanol, or ethyl acetate.

8. The determination method according to claim 1, characterized in that, The absorbance was measured at 484 nm.

9. The determination method according to claim 1, characterized in that, The degradation rate is the ratio of the absorbance value of the supernatant of the degradation group to the absorbance value of the supernatant of the total sugar group.

10. The application of the assay method according to any one of claims 1 to 9 in drug delivery, tissue engineering, and wound dressing development.

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